Cardiomyocytes and methods of producing and purifying cardiomyocytes

ABSTRACT

The invention provides methods for producing a culture of cardiomyocytes and cultures of cardiomyocytes. Exemplary methods of producing and cultures of cardiomyocytes include a population of cells including cells having spontaneous and periodic electrical activity, and/or including nodal, sino-atrial or pacemaker cells; immature cardiomyocytes (cardiomyoblasts); mature contractile cardiomyocytes; or a mixed population of two or more of such cells.

RELATED APPLICATIONS

This application claims the benefit or priority of U.S. application Ser.No. 60/895,066, filed Mar. 15, 2007, and is expressly incorporatedherein by reference.

INTRODUCTION

1. Myocardic Development

As the embryo is growing thicker than 300-400 microns, the diffusion ofnutrients, oxygen, and carbon dioxide is inadequate. A circulatorysystem is needed and developed by the middle of the fourth week ofgestation (embryonic day 24). Heart and skeletal muscle progenitor cellsare derived from distinct regions of the mesoderm: lateral platemesoderm and paraxial mesoderm, respectively. Cells from the cranialparaxial mesoderm (anterior splanchnic mesoderm) contribute to bothmyocardial and endocardial cell populations. The primordium of the heartforms in the cardiogenic plate located at the cranial end of the embryo.

2. Development of the Conduction System

The factor that directs myocardial development toward either a workingmyocardium phenotype or a conduction system phenotype is not known. Onthe basis of positional clues, neural crest cells might play a role inthe induction of the central conduction system and for theepicardium-derived cells in differentiation of the Purkinje network.These neural crest cells present as a central mass of condensedmesenchyme between the fourth and sixth pharyngeal arch arteries andextend two prongs deep down into the proximal cushions. This phenomenonwas first described in the avian embryo and confirmed in human embryosduring the fusion of the cushions from distal to proximal.

3. Growth Factors and Pathways Involved in Cardiac Development

Bone morphogenic protein 4 and 2 (BMP4, BMP2) induces cardiacdifferentiation in the cranial paraxial mesoderm, and blocks thedifferentiation of skeletal muscle precursors in these cells. Bonemorphogenic proteins belong to the transforming growth factor beta superfamily (TGF-beta). Ligand binding to its receptor induces the formationof a complex in which the Type II BMP receptor phosphorylates andactivates the Type I BMP receptor. The Type I BMP receptor thenpropagates the signal by phosphorylating a family of signal transducers,the Smad proteins. Currently, eight Smad proteins have been cloned (Smad1-7 and Smad 9). Upon phosphorylation by the BMP Type I receptor, Smad1can interact with either Smad4 or Smad6. The Smad1-Smad6 complex isinactive; however, the Smad1-Smad4 complex triggers the expression ofBMP responsive genes. The ratio between Smad4 and Smad6 in the cell canmodulate the strength of the signal transduced by BMP. BMP signaling isregulated at different molecular levels, most important—Noggin and othercystine knot-containing BMP antagonists bind with BMP-2, 4 and 7 andblock BMP signaling. If activated, Smad6 binds type I BMP receptor andprevents Smad1, 5 and 8 to be activated. The BMP family comprises: BMP1,BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15. The mostimportant factors involved in the cardiac development are BMP2, BMP4,BMP7 and BMP10.

Wnt-mediated signals from the underlying neural tube and notochordsuppress cardiomyocyte specification. Wnt proteins form a family ofhighly conserved secreted signaling molecules that regulate cell-to-cellinteractions during embryogenesis. Wnt proteins bind to receptors of theFrizzled and LRP families on the cell surface. Through severalcytoplasmic relay components, the signal is transduced to b-catenin,which then enters the nucleus and forms a complex with TCF to activatetranscription of Wnt target genes. To date, there are at least 19 knownWnts in human, spread over 10 chromosomes. The Wnts involved inmesodermal and cardiac development are: Wnt-2b/13; Wnt-3/Int-4; Wnt-3a;Wnt-8a/8d.

Inhibitors of Wnt family (Crescent, Dkk-1, and glycogen synthasekinase-3) determine a gradient along the cardiogenic area (lateralmesoderm and anterior endoderm). Administration of exogenous crescent orDkk-1 to posterior lateral plate mesoderm induces heart muscle formationwhile repressing erythropoiesis. Conversely, ectopic expression ofeither Wnt-8c or Wnt-3a in precardiac mesoderm blocks cardiogenesis inthis tissue while promoting formation of primitive erythrocytes.

Cripto signaling primes differentiation of embryonic stem cells intocardiomyocytes and inhibits neural differentiation of ES cells. Criptois a member of the CFC-EGF family of both secreted and membrane anchoredproteins. Cripto mediates the binding of Noda1, Vg1, and GDF1 to theactivin receptors. Failure to activate Cripto signaling results inconversion of ES cells into a neural fate. EGF-CFC domain (epidermalgrowth factor-Cripto/FRL-1/Cryptic coreceptors) alone is sufficient forCripto activity in the cardiogenic induction.

TGFβs, Activin, Noda1, and Vg1/GDF1 are ligands for EGF-CFC coreceptorsand potent mesendoderm inducers in vertebrates.

Endothelin-1 (ET-1) is an endothelium-derived peptide with potentvasoconstrictor and proliferative properties. It is involved in theconduction system development.

The gradient of BMP (BMP2, BMP4, BMP7) signaling can be regulated withligand traps: DAN, Cerberus, Chordin/SOG, Follistatin, Noggin.

Markers for myocardic development

Nkx2.5/Csx is first expressed in the presumptive precardiac mesoderm,and is later restricted to the bilateral dorsal regions that willdevelop into the muscular portions of the heart, which is maintainedthroughout development. The number of cells in the cardiac conductionsystem is directly related to Nkx2-5 gene dosage. Nkx2.5 mutations areassociated with conduction system abnormalities in both mouse and man.Tanscription factors that are expressed during cardiac developmentinclude, for example, a GATA binding family transcription factor (GATAbinding protein, such as GATA 4), MEF2 (Myocyte Enhancer Factor 2), HAND(heart and neural crest derivatives), Irx, Tbx, and HRT families oftranscription factors. Additional factors include, for example, SRF(serum response factor), Isl1 (Islet1), LIM (named from the Lin-11,Isl-1 and Mec-3 genes) and alpha-actin.

SUMMARY

The invention provides methods for producing a culture ofcardiomyocytes. In one embodiment, a method includes: providing aculture of stem cells that are at least 90% confluent or the cells haveovergrown to form multiple layers of cells, or proliferating stem cellsuntil the cells are at least 90% confluent or the cells have overgrownto form multiple layers of cells; inducing formation of mesoderm bycontacting the overgrown stem cells with a bone morphogenic protein(BMP) receptor ligand and an fibroblast growth factor (FGF) receptorligand for a period of about 2 to 15 days; and promoting cardiomyocyteformation by contacting mesoderm cells with neural cells or endodermcells, or a neural cell or endoderm cell conditioned culturesupernatant, for a period of about 1 to 21 days, thereby producing aculture of cardiomyocytes. In particular methods embodiments,cardiomyocytes comprise a population of cells including cells havingspontaneous and periodic electrical activity, and/or including nodal,sino-atrial or pacemaker cells; immature cardiomyocytes(cardiomyoblasts); mature contractile cardiomyocytes; or a mixedpopulation of two or more of such cells.

The invention also provides cardiomyocytes at any developmental,maturation or differentiation stage. Such cells are indicated at thevarious time points in Table 1, and can represent a mixed population ofcells at a particular developmental, maturation or differentiationstages or a relatively uniform population of cells in which a majorityof cells (e.g., 50%, 60%, 70%, 80%, 90% or more) is at a particulardevelopmental or maturation type or stage. Such cells therefore includecells having spontaneous and periodic electrical activity, and/orincluding nodal, sino-atrial or pacemaker cells; immature cardiomyocytes(cardiomyoblasts); mature contractile cardiomyocytes; or a mixedpopulation of two or more of such cells. Such cells include progenitorcells and precursor cells of the cells indicated at the various timepoints in Table 1, as well as daughter cells arising from the cellsindicated at the various time points in Table 1. In particularembodiments, a cell culture includes immature cardiomyocytes(cardiomyblasts), wherein 50% or more (e.g., 60%, 70%, 80%, 90%, etc.)of said culture comprises immature cardiomyocyte (cardiomyblast) cells.In additional particular embodiments, a cell culture includes immaturecardiomyocytes (cardiomyblasts) in which no more than about 30% (e.g.,no more than about 25%, 20%, 15%, 10%, 5%, etc.) of said cells beat orcontract.

DRAWING DESCRIPTIONS

FIG. 1 shows a flowchart of cardiomyocyte production and purification.

FIGS. 2A-2C show A) High density stem cell culture prior induction; B)cardiomyocyte colony, centered by nodal tissue and surrounded by lessdifferentiated mesodermal population ; and C) cardiomyocytes organizedin syncytial trabeculy. Less differentiated cardioblasts between theorganized cardiomyocytes.

FIGS. 3A-3N show cardiomyoblasts after growth under certain conditionsfor certain periods of time: A) Homogenous growth of cardiomyoblasts ina 18 day old culture showing formation pf trabecular clusters; B)syncitial structures (arrows) after 21-24 days of differentiation; C)cardiomyocyte cultures can be dissociated and replated on adherentsubstrate at earlier stages, when NKX2.5 transcription factor ispresent; D) Nuclear staining with bisbenzimide; E) merged pictures; F)mature myocardial cells fuse and form trabecular structures positive fora-actin; G) bisbenzimide nuclear stain; H) merged picture. (60×); I)actin labeling persists after enzymatic dissociation of the cultures; J)nuclear stain with bisbenzimide; K) Merged picture (20×); L) and M)Colabeling with NKX2.5 (L) and actin (M), shows the overall expressionof NKX2.5, while the actin is progressively expressed; N) Nuclearcounterstain (100×).

DETAILED DESCRIPTION

Expansion of stem cells is important in early stage since priming canaffect outcome. Priming is a slight tendency of the stem cell culture todifferentiate towards ecto- or endoderm. Since cardiac tissue has amesodermal origin, a good balance between ectoderm and endodermpopulations is indicative of successful differentiation. A healthystroma, defined as the cells resulting from spontaneous differentiationin an ES cell culture, is a good differentiation prognostic. Thefibroblastic shaped cells surrounding the colonies typically have clearcytoplasm without vacuoles, little or nor membrane fragmentation andlittle or no tendency for detachment from substrate. An ectodermdominance can be recognized if adjacent to stem cell colonies extensiveepithelial plaques or small triangular cells are observed. An endodermdominance is shown by polygonal cell with abundant cytoplasm, mostlygrowing in the center of the colonies.

Induction creates conditions in which differentiation of stem cells ispushed toward mesoderm. Balancing the FGF/Wnt signals with the oppositeBMP favors the development of cardiac tissue. Induction can beaccomplished in various ways.

Exemplary physical conditions include growth of stem cell cultures tohigh density, hypoxia and acidosis. One way is to grow the cultureswithout splitting over the passage point, e.g., with over 90% or more,or 100% confluency. Non discriminative counts (including the stem cellsand stromal cells) are typically greater than about 150,000 cells/cm2 to250,000 cells/cm2. The stroma appears dense, and the colonies tend toform multilayers, as observed by a rough, yellow color on top of thecells. Following stem cell over growth, acidosis and hypoxic conditionswill develop in the culture. A pH decrease alternatively can beaccomplished optionally using organic acids (e.g., lactic, citric,fumaric, ascorbic, folic, malic, succinic, oxalacetic, ketoglutaricacids, etc.) to a pH value of not less than about 6.5-6.8. Hypoxia canbe accomplished in incubators with a low oxygen concentration (0.5 to2%) and a deeper supernatant (5 mm to 10 mm)(recommended). The media canthen be replaced with a new formulation. Appropriate basal mediainclude. For example, MEM, DMEM, F12 and mixtures thereof Optionalsupplements include, for example, glucose, B27 supplement, albumin(e.g., human), essential and non essential aminoacids, Glutamax 1× orL-Glutamine 1×, a thyroid hormone (e.g., T3/4, 20 ng/ml), insulin (e.g.,10 ug/ml), transferrin, ethanolamine, sodium selenite, a hydrosolublevitamin, and a liposoluble vitamin.

Signaling can be used to induce cardiomyogenesis. Exemplary signaling istwo or more signals used to induce cardiomyogenesis, for example, aligand for FGF receptor and a ligand for BMP receptor. The signals canbe added to the culture from exogenous sources or secreted by cocultureor supernatant from specialized cells. Ligands for the FGF receptorinclude, for example, FGF basic (FGF2, bFGF). Other members of the FGFfamily can be used (for example FGF1). The amount the FGF is typicallyabout 5 to 20 ng/ml but can be extended from 2 to 200 ng/ml. Ligands forthe BMP receptors include, for example, BMP4. Other BMPs can be used,for example BMP2, BMP7 or a combination. The BMPs are typically used ata concentration of about 0.5 to 10 ng/ml, but can be from 0.1 to 100ng/ml.

A Wnt family member may also be optionally used in conjunction withabove factors in cell cultures. One example of a Wnt family member isWnt-5a (e.g., at a concentration of 1 to10 ng/ml).

Induction is accomplished by exposing the stem cells to one or more ofthe above physical conditions and signaling factors for 3 to 10 days,typically for about 7 days. During induction, growth conditions(hypoxia, acidosis) will cause extensive cellular loss. Inhibition orprevention of cell death (protection) can be accomplished using selenium(for example, sodium selenite) at a concentration of about 1 to 20 ng/ml(0.5 to 50 ng/ml), lithium (for example, lithium carbonate) at aconcentration of about 1-20 ng/ml (0.5-50 ng/ml); ascorbic acid orascorbate (for example sodium or calcium ascorbate) 1 ug/ml (0.1 to 20ug/ml); superoxide dismutase (SOD) 1-10 u/ml (1-100 u/ml). These andother protective elements can be used in combination, for increasedefficacy. Each individual component is optional; typically a combinationof two is used.

After induction to promote development of cardiomyocytes, for about thenext 10-14 days, signaling which promotes the cardiomyocyte developmentcan be maintained by exposing the cell culture to other cell populationsknown as a source of signal factors. This cell population can be placedin the same culture dish or cultured separately and the supernatantmedia used to feed the cardiac cells. The other cell populations thatcan be used include neural cells, which can spontaneously or be directedto differentiate in the same culture, for example, by addition ofretinoic acid (e.g., 1-20 mMol or 5-10 mMol). Alternatively,cardiomyocyte media can be incubated overnight (e.g., 12-48, or about 24hours) on pre-differentiated or primary neural cultures or neuralprogenitor cultures. Endodermal cells can develop in the same culturedish with the cardiomyocytes after exposure to BMPs. Alternatively, aseparate culture of endodermal cells from differentiated embryonic stemcell or a primary culture of endo-epithelial cells could also be used tocondition the cardiomyocyte media for a period of time (e.g., 12-48, orabout 24 hours).

During promotion of cardiomyogenesis other factors which stimulatecellular metabolism, division and growth (proliferation) can be added toenrich (increase numbers) the cardiomyocyte population. Non limitingexamples include insulin (e.g., 5 to 50 μg/ml, 15 μg/ml), thyroidhormones (e.g., T3/4, 1 to 40 ng/ml, 20 ng/ml) and IGF (e.g., 5-50ng/ml, 10 ng/ml).

After about 21 to 28 days of growth, beating (contractile) cellularconglomerates are typically observed. Beating (contractile) frequencyvaries with the media pH, temperature, and responds to variouscardiomodulator drugs (e.g., catecholamine, calcium blockers, potassium)

Contracting (beating) cardiomyocytes tend to terminally differentiateand undergo cell cycle arrest. A cardioplegic solution or treatment canprotect cardiomyocytes by inducing a rapid diastolic arrest, minimizingenergy requirements and inhibiting or preventing hypoxic damage duringpurification. A mixture which includes a membrane stabilizer and/or a Cachannel inhibitor is an example of a cardioplegic solution, which can beadded as needed (e.g., daily to feeding media). Particular components ofsuch a mixture include KCl (e.g., 10-20 mM), MgCl₂ (e.g., 10 mM), CaCl₂(e.g., 1.2 mM), puerarin (e.g., 0.5 mM), Nifedipine (a calcium channelinhibitor that prevents intracellular Ca accumulation, e.g., 1-10 u/ML),tromethamine (e.g., Tris) buffer (e.g., 0.3 M) adjusted to a pH of about8.6, L-Monosodium Glutamate Monohydrate, L-Monosodium AspartateMonohydrate or a mixture of L-Monosodium Glutamate Monohydrate (e.g.,4.277%), L-Monosodium Aspartate Monohydrate (e.g., 3.923%). Thecomponents are mixed, sterile filtered and typically used in 5% to 50%v/v dilution with growth media.

For purification, one optional method for selection of cardiomyocytes isby identification of beating colonies. With the use of an ablator laser(XY Clone, Zeiss etc) or manually surrounding cells or the colonies areremoved in separate dishes. Another method uses the ability of thecardiomyocyte to switch to anaerobic metabolism, while the other cellsin the culture that aerobically metabolize are killed. In particular,for example, exposure to absolute hypoxia and exposure to lactic acid incardioplegic solution for about 30-60 minutes.

After purification, cells can be recovered using media enriched withcardioplegic mixture and dissociated and re-plated in new dishes coatedwith an adherent substrate to expand cells. Hyperplasia can follow forabout another week and purification can be repeated before dissociation.If the cardioplegic mixture is removed from media, the cardiomyocytesshould restart contractile (beating) activity shortly.

For preservation in diastolic arrest, cardiomyocytes can becryopreserved and stored indefinitely in liquid nitrogen. Freezing canbe done after dissociation, or with cells attached to culture plates. Ifintended for transportation involving a limited time (e.g., 1-12 hours),diastolic arrest in conjunction with hypothermia (e.g., +4 C to +30 C)can be employed without cryopreservation.

Cardiomyocytes, at different developmental stages, can be used forvarious applications. Two particular applications are in vitro drugtesting and in vivo cell replacement therapy. Mature cardiomyocytes withsyncytial morphology and contractile activity are suitable for in vitrodrug testing or screening. These cells tolerate fewer manipulations andare recommended not to be dissociated.

Cardioblasts, immature cardiomyocytes can be used for transplant ascellular replacement therapy. Such cells can be collected beginning fromthe promotion phase of differentiation, and stored frozen aftercryopreservation. Atrial/pacemaker cells from the central areas of thecardiomyocyte colonies, can be used for pacemaker cellular replacementin vivo.

Table 1 provides an overview of conditions, media, cardioplegictreatment, factors and other components, cell markers and morphologicfeatures at the various stages of cardiomyocyte production,characterization and isolation. Populations of cells at any of thevarious time points in Table 1 can be isolated, purified or expanded, orprovided as a cell culture or kit or other composition. The inventiontherefore provides isolated and purified cells at each of the varioustime points, as well as a cell culture or kit or other composition thatincludes cells at each of the various time points, as well as daughtercells derived therefrom (undergo subsequent proliferation, maturation,development or differentiation into additional cells).

Populations of cells at various time points in Table 1 can represent amixed population of cells at different developmental, maturation ordifferentiation stages or a relatively uniform population of cells inwhich a majority of cells (e.g., 50%, 60%, 70%, 80%, 90% or more) is ata particular developmental or maturation type or stage. Populations ofcells at various time points in Table 1 can also be maintained at thatstage, in other words, development or maturation of one or more celltypes within the population generally is arrested at a particular stage,such that the cells remain in at the stage (e.g., cardioblasts). Suchcells can be expanded (proliferate) while maintaining the cells at aparticular developmental, maturation or differentiation stage, or can bereleased (unlocked) to mature or further develop or differentiate, inaccordance with the invention.

TABLE 1 Prerequisite Induction Promotion Hyperplasia Purification Time0-3 days 7 days 14 days 7 days 1-2 day Physical Attached, Attached tosubstrate, Attached Attached Feeding Attached on substrate conditionsmesodermal Hyperconfluency, Feeding accordingly to Hypoxia <2% for 30-60min stroma. No 100%-150%, Cellular accordingly to cellular density,tendencies toward stress: deep media 5-20 mm, cellular density, reducedcellular ecto or endoderm hypoxia reduced cellular stressdifferentiation (Oxygen <5-2%) and stress acidosis (pH 7.0-7.2) MediaStem cell media Cardio media Cardio media Cardio media + Cardio media +composition Cardioplegic Cardioplegic mixture (when mixture +contractile colonies Lactic acid (for 30-60 min) observed) Protection NoSelenite, Lithium, Selenite Selenite After 30-60 min of Ascorbate,Lithium Lithium exposure: Selenite Superoxyde SOD, SOD, LithiumDismutase (SOD), bMercaptoEthanol bMercaptoEthanol SOD, bMercaptoEthanolbMercaptoEthanol Growth FGF 10 ng/ml FGF 10 ng/ml BMP4 FGF 2 (and/or FGF2 (and/or After recovery FGF 2 factors (and/or 2, 7) 1-5 ng/ml FGF1) -5-10 ng/ml FGF1) - 5-10 ng/ml (and/or FGF1) - 5-10 ng/ml BMP4 (and/or 2,7) 1-5 ng/ml Optional Organic acids: lactic, Media incubated Mediaincubated Piruvic acid components citric, ascorbic, over night onovernight on neural Ascorbic acid piruvic. neural cultures if culturesif RA not Thyroid hormones: 5-10 Concentration RA not used at used atinduction ng/ml adjusted to media pH induction (separate Noggin 1-20ng/ml IGF 10 ng/ml not lower than 6.8 neural exposure) alternative to RA(10 uM) for 3 Noggin 1-20 ng/ml neural exposure days if mixed neuralalternative to Thyroid horm: 5-10 ng/ml exposure is decided neuralexposure IGF 10 ng/ml Thyroid hormones: 5-10 ng/ml IGF 10 ng/mlMacroscopic Delimited colonies, Layering observed in Dense cellular Incardioplegic The cells surrounding characterization with fibroblastichyperdense culture, agglomerations solution no beating the cardiomyocytestroma media with phenol with prominent colonies should be coloniesshould be red colored yellow center observed and the progressivelybefore feeding. (umbilicated). colonies should eliminated Coloniesdelimited Toward the end of progressively poorly, mesodermal the period,the increase in size transformation of the cellular stem cellsagglomerations will initiate spontaneous beating Markers Oct4, SSEA4,Nkx2.5, MEF2, + a-actin, + a-actin, Tra1-81, PA HAND, Isl1, LIMultrastructure ultrastructure

In accordance with the invention, methods for producing a cell cultureof cardiomyocytes are provided. In one embodiment, a method includescontacting stem cells with a bone morphogenic protein (BMP) receptorligand and a fibroblast growth factor (FGF) receptor ligand for a periodof about 2 to 15 days to induce mesoderm cells; and contacting mesodermcells with neural cells or endoderm cells, or a neural cell or endodermcell conditioned culture supernatant, for a period of about 1 to 21days, thereby producing a cell culture of cardiomyocytes. In anotherembodiment, a method includes providing a culture of overgrown stemcells or proliferating stem cells until the cells are overgrown;contacting the overgrown stem cells with a bone morphogenic protein(BMP) receptor ligand and an fibroblast growth factor (FGF) receptorligand for a period of about 2 to 15 days to induce formation ofmesoderm; and contacting mesoderm cells with neural cells or endodermcells, or a neural cell or endoderm cell conditioned culturesupernatant, for a period of about 1 to 21 days, thereby producing aculture of cardiomyocytes.

Cardiomyocytes in one aspect, comprise a population of cells havingspontaneous and periodic electrical activity. Cardiomyocytes in anotheraspect, include nodal, sino-atrial or pacemaker cells; immaturecardiomyocytes (cardiomyoblasts); mature contractile cardiomyocytes; ora mixed population thereof. Such populations of cardiomyocytes can bemaintained for a period of time (e.g., 1-24 minutes, hours, days, weeks,etc.) at a given developmental, maturation or differentiation stage, canbe expanded or can be allowed to progress to a subsequent developmental,maturation or differentiation stage.

Stem cells of the methods and compositions of the invention includecells, such as cells of a cell monolayer with greater than about 60%,70%, 80%, 90%-95% or more confluency (e.g., 96%, 97%, 98%, etc. . . .100%). Overgrown cells are cell cultures greater than 100% confluencythat form multiple layers of cells. Stem cells of the methods andcompositions of the invention also include overgrown cells, such ascells that form a multilayer culture. Stem cells of the methods andcompositions of the invention also include overgrown cells, such ascells having a density of about 150,000 to about 250,000 cells/cm².

Methods for producing a cell culture of cardiomyocytes, as well as cellpopulations of cardiomyocytes include addition of an acid. Exemplarynon-limiting examples include lactic, citric, fumaric, ascorbic, folic,malic, succinic, oxalacetic, or ketoglutaric acid.

Methods for producing a cell culture of cardiomyocytes, as well as cellpopulations of cardiomyocytes include subjecting mesoderm cells tohypoxia, or having a cell population of cardiomyocytes which have beensubjected to hypoxia. In particular aspects, the stem cells, mesodermcells or cardiomyocytes are subjected to or are contained or comprisedwithin an oxygen concentration of about 0 to 2.0%, such as cell culturesincluding cardiomyocytes in which such cells have been treated orsubjected to hypoxia (e.g., 0%-2% oxygen). Degassing of growth medium toreduce or remove ambient or dissolved oxygen is typically performed whencells are subjected to hypoxia or treated with an agent that induces orstimulates hypoxia.

Methods for producing a cell culture of cardiomyocytes, as well as cellpopulations of cardiomyocytes further include addition of an agent,treatment or condition that inhibits cell death or apoptosis. In variousaspects, an agent includes selenium (e.g., sodium selenite), lithium(e.g., lithium carbonate); ascorbic acid or ascorbate (e.g., sodium orcalcium ascorbate) or superoxide dismutase (SOD). Such agents,treatments and conditions can reduce or protect cardiomyocytes andpopulations of cardiomyocytes from cell death or apoptosis. Such agentsmay be at an effective concentration, for example, selenium at aconcentration of 1 to 20 ng/ml, or 0.5 to 50 ng/ml; lithium at aconcentration of 1 to 20 ng/ml, or 0.5-50 ng/ml; ascorbic acid orascorbate at a concentration of 0.1 to 20 ug/ml, or 1 ug/ml; orsuperoxide dismutase (SOD) at a concentration of 1 to 100 u/ml, or 1 to10 u/ml.

Methods for producing a cell culture of cardiomyocytes can includecontacting mesoderm cells with a wingless-int-1 (Wnt) family member. Invarious aspects, a wingless-int-1 (Wnt) family member is Wnt-5a.Wingless-int-1 (Wnt) family members can be at an effectiveconcentration, for example, Wnt-5a at a concentration of about 1 to 10ng/ml.

Methods for producing a cell culture of cardiomyocytes, as well as cellpopulations of cardiomyocytes include expression of a marker associatedwith cardiomyocytes, at a given deleopmental, maturation ordifferentiation stage. Such cells may be progenitor or precursor cellsof cardiomyocytes. In one embodiment, at least a portion of cellsexpress Nkx2.5/Csx marker. In additional embodiments, at least a portionof cells express a GATA binding family transcription factor (GATAbinding protein, such as GATA 4), MEF2 (Myocyte Enhancer Factor 2), HAND(heart and neural crest derivatives), Irx, Tbx, or HRT family oftranscription factors, SRF (serum response factor), Isl1 (Islet1), LIM(named from the Lin-11, Isl-1 and Mec-3 genes) or alpha-actin marker.

Neural cells can be produced within the culture of cardiomyocytes or canbe provided to the culture of cardiomyocytes by way of a separateculture of neural cells (e.g., pre-differentiated or primary neuralcells or neural progenitor cells). In one embodiment, neural cells canbe produced by addition of a retinoic acid receptor ligand to mesodermcells. Exemplary non-limiting retinoic acid receptor ligand includesretinoic acid, for example, a concentration of 1-20 mMol, or 5-10 mMolretinoic acid. In another embodiment, cardiomyocyte formation ispromoted by contact with neural cells for a period of time, for example,about 12-48 hours.

Endoderm cells can be produced within the culture of cardiomyocytes orcan be provided to the culture of cardiomyocytes by way of a separateculture of endoderm cells (e.g. primary culture of endo-epithelialcells). In one embodiment, endoderm cells can be produced by addition ofa BMP receptor ligand to mesoderm cells. In another embodiment,cardiomyocyte formation is promoted by contact with endoderm cells for aperiod of time, for example, about 12-48 hours.

Methods for producing a cell culture of cardiomyocytes can includecontacting mesoderm cells with insulin; a thyroid hormone, or aninsulin-like growth factor (IGF). Exemplary non-limiting insulin is aconcentration of about 5 to 50 μg/ml, or about 15 μg/ml. Exemplarynon-limiting thyroid hormone is a concentration of about 1 to 40 ng/ml,or about 20 ng/ml. A particular thyroid hormone includes T3/4. Exemplarynon-limiting IGF is a concentration of about 5 to 50 ng/ml, or about 10ng/ml. A particular IGF is IGF-1.

Exemplary non-limiting examples of FGF receptor ligand include FGF basic(FGF2, bFGF), acidic FGF (FGF1, aFGF), and combinations thereof.Exemplary non-limiting amounts (concentrations) of FGF is between about2 to 200 ng/ml, or about 5 to 20 ng/ml.

Exemplary non-limiting examples of BMP receptor ligand include BMP4,BMP2, BMP7), and combinations thereof. Exemplary non-limiting amounts(concentrations) of BMP receptor ligand is between about 0.1 to 100ng/ml, or about 0.5 to 10 ng/ml.

Methods for producing a cell culture of cardiomyocytes, as well as cellpopulations of cardiomyocytes include growth medium, which can be addedor changed at any time, for a period of 1-60 minutes, 1-60 hours or 1-60days. In exemplary embodiments, fresh growth media is added prior to,during or following a step of a method of the invention. In additionalexemplary embodiments, fresh growth media is added to a cell culture ofthe invention at a given developmental, maturation or differentiationstage, or during cell expansion (proliferation).

Exemplary cell media for producing a cell culture of cardiomyocytes, aswell as cell populations of cardiomyocytes include a stem cell media ora cardio cell media. Stem cell or a cardio cell media can include abasal media and one or more of supplements, such as human albumin,essential amino acids, non essential amino acids, L-glutamine, a thyroidhormone, insulin, transferrin, ethanolamine, sodium selenite, ahydrosoluble vitamin, a liposoluble vitamin or B27 supplement. Exemplarybasal media includes DMEM, F12 or DMEM:F12 (e.g., in a ratio of about11).

Methods for producing a cell culture of cardiomyocytes, as well aspopulations of cardiomyocytes that include non-beating contractilecardiomyoblasts are provided. Methods for producing a cell culture ofcardiomyocytes, as well as populations of cardiomyocytes that includebeating contractile cardiomyocytes are provided. Relative proportions oramounts of such cell types within cell cultures include 50%, 60%, 70%,80%, 90% or more non-beating contractile cardiomyoblasts in a cellculture, as well as 50%, 60%, 70%, 80%, 90% or more beating contractilecardiomyocytes in a cell culture.

Beating (contractile) frequency of cardiomyocytes can be modulated byculture media pH, temperature, or a modulator drug. Exemplarynon-limiting modulator drugs include catecholamine, a calcium channelblocker, or potassium.

Methods for producing a cell culture of cardiomyocytes, as well aspopulations of cardiomyocytes include contacting cardiomyocytes orcontact of cardiomyocytes with a cardioplegic solution or treatment, andcardiomyocytes in a cardioplegic solution or treatment. Exemplarynon-limiting cardioplegic solution or treatment (e.g., hypoxia) inducesdiastolic arrest of beating cardiomyocytes. Exemplary non-limitingcardioplegic solution or treatment (e.g., hypoxia) reduces energyrequirement of beating cardiomyocytes. Exemplary non-limitingcardioplegic solution or treatment (e.g., hypoxia) inhibits hypoxiainduced damage of contractile (beating) cardiomyocytes. Non-limitingcardioplegic solution includes a mixture of one or more of: KCl (10-20mM); MgCl₂ (10 mM); CaCl2 (1.2 mM); puerarin (0.5 mM), or Nifedipine(1-10 uM). Non-limiting cardioplegic solution can also include atris(hydroxymethyl)aminomethane or Hanks balanced salt solution adjustedto a pH of approximately 8.6). Non-limiting cardioplegic solution canfurther include a one or more of of L-Monosodium Glutamate Monohydrateor L-Monosodium Aspartate Monohydrate, or a mixture of L-MonosodiumGlutamate Monohydrate and L-Monosodium Aspartate Monohydrate, forexample, about 3-5% (e.g., 4.277%) of L-Monosodium Glutamate Monohydratewith or without about 3-5% (e.g., 3.923%) of L-Monosodium AspartateMonohydrate.

Methods for producing a cell culture of cardiomyocytes, as well aspopulations of cardiomyocytes that include isolating and isolatedimmature cardiomyocytes (cardioblasts) prior to beating (contractileactivity) are provided. Methods for producing a cell culture ofcardiomyocytes, as well as populations of cardiomyocytes that furtherinclude isolating and isolated mature contractile (beating)cardiomyocytes are also provided.

Methods for producing a cell culture of cardiomyocytes, as well aspopulations of cardiomyocytes include preserving and preservedcardiomyocytes. In various embodiments, preserving and preservedcardiomyocytes include freezing (frozen) or storing (stored)cardiomyocytes, such as, for example, cells having spontaneous andperiodic electrical activity; immature cardiomyocytes (cardioblasts);nodal, sino-atrial or pacemaker cells; mature contractilecardiomyocytes; and mixed populations thereof.

Methods for producing a cell culture of cardiomyocytes, as well aspopulations of cardiomyocytes that include enriching and enriched,selecting and selected cardiomyocytes are provided. Cell cultures andmethods for producing cell cultures by such methods include cellsproduced by a treatment that requires anaerobic metabolism so that cellsunable to survive by anaerobic metabolism senesce or die are provided,thereby enriching for cells that survive via anaerobic metabolism. Cellcultures and methods for producing cell cultures by such methods includeconditions of reduced oxygen (e.g., less than 2%), such as hypoxia,contact with lactic acid or contact with a cardioplegic solution ortreatment.

Methods for producing a cell culture of cardiomyocytes, as well aspopulations of cardiomyocytes include enriching (enriched) or selecting(selected) cardiomyocytes of a selected developmental, maturation ordifferentiation stage are provided. In various non-limiting embodiments,a cardiomyocyte population includes nodal, sino-atrial or pacemakercells, mature contractile cardiomyocytes, immature cardiomyocytes(cardioblasts), or a mixed population thereof. Cardiomyocytes ofappropriate developmental, maturation or differentiation stage can beidentified and removed or recovered. Recovering (recovered) or removingenriched or selected cardiomyocytes of appropriate developmental,maturation or differentiation stage are therefore included in theinvention. Enriched or selected cardiomyocytes recovered or removed canbe distributed in a cell culture dish, plate, vial, tube, flask orbottle. Enriched or selected cardiomyocytes that are recovered orremoved can be frozen, for example, at −20 degrees C. or less, e.g., −70degrees C.

In particular embodiments, a cell culture includes immaturecardiomyocytes (cardiomyblasts), wherein 50% or more (e.g., 60%, 70%,80%, 90%, etc.) of said culture comprises immature cardiomyocyte(cardiomyblast) cells. In additional particular embodiments, a cellculture includes immature cardiomyocytes (cardiomyblasts) in which nomore than about 30% (e.g., no more than about 25%, 20%, 15%, 10%, 5%,etc.) of said cells beat or contract. In further particular embodiments,a cell culture includes a population of cells that are able to surviveanaerobic conditions (e.g., an atmosphere of 0%-2% oxygen) for a periodof time (e.g., 1-60 minutes or 1-8 hours). In a particular aspect, 75%or more of cardiomyocyte (e.g., immature cardiomyocyte (cardiomyblast))cells survive anaerobic conditions for 30, 60 or more minutes. Inanother particular aspect, no more than about 25% (no more than about20%, 15%, 10%, 5%, etc.) of cardiomyocyte (e.g., immature cardiomyocyte(cardiomyblast)) cells die when subjected to anaerobic conditions for30, 60 or more minutes. Such embodiments include cell cultures ofimmature cardiomyocytes (cardiomyblasts) in which the immaturecardiomyocyte (cardiomyblast) cells proliferate. Such embodimentsfurther include cell cultures of immature cardiomyocytes(cardiomyblasts) in which no more than about 30% (no more than about25%, 20%, 15%, 10%, 5%, etc.) of said immature cardiomyocyte(cardiomyblast) cells exhibit a function associated with contractile(beating) cardiomyocytes.

Cell cultures can be included in a container, or attached to asubstrate. In a particular embodiment, a cell culture includes asubstrate to which the cells are attached. Exemplary containers andsubstrates include tissue culture dishes or plates, glass or plasticslides, multiwell plates or dishes having disposed therein thecardiomyocyte (cardiomyblast) cells in one or more of said wells.

Cardiomyocytes, as well as populations of cardiomyocytes includingenriched or selected cardiomyocytes of any developmental, maturation ordifferentiation stage thereof can be used to screen for or identifycardioactive agents. In various non-limiting embodiments, acardiomyocyte population used in a screen or identification methodincludes nodal, sino-atrial or pacemaker cells, mature contractilecardiomyocytes, immature cardiomyocytes (cardioblasts), or a mixedpopulation thereof.

In accordance with the invention, there are provided methods ofscreening and identifying cardioactive agents. In one embodiment, amethod includes contacting a cardiomyocyte with a test agent; anddetermining if the test agent modulates an activity or function ofcardiomyocytes within the population. A test agent modulating anactivity or function of cardiomyocytes within the population identifiesthe test agent as a cardioactive agent. Exemplary activity or functionthat can be modulated include contraction or beating, or production of ametabolic product (e.g., production of one or more of urea, creatine orCO2), or intracellular enzyme (e.g., one or more of lactatedehydrogenase, creatine phosphokinase (CPK), creatine kinase (CK) ortroponin), or cellular apoptosis, necrosis, death; orde-differentiation, maturation, or division.

Methods of screening and identifying cardioactive agents include thosesuitable for high throughput screening, which include arrays ofcardiomyocyte cells (e.g., microarrays) positioned or placed, optionallyat pre-determined locations or addresses. High-throughput robotic ormanual handling methods can probe chemical interactions and determinelevels of expression of many genes in a short period of time. Techniqueshave been developed that utilize molecular signals (e.g., fluorophores)and automated analyses that process information at a very rapid rate(see, e.g., Pinhasov et al., Comb. Chem. High Throughput Screen. 7:133(2004)). For example, microarray technology has been extensivelyutilized to probe the interactions of thousands of genes at once, whileproviding information for specific genes (see, e.g., Mocellin and Rossi,Adv. Exp. Med. Biol. 593:19 (2007)).

Such high-throughput screening methods can identify cardioactive agents.For example, cardiomyocyte cells (e.g., cardiomyoblasts, cardiomyocytesor sino-atrial nodal cells) can be positioned or placed (pre-seeded) ona culture dish, tube, flask, roller bottle or plate (e.g., a singlemulti-well plate or dish such as an 8, 16, 32, 64, 96, 384 and 1536multi-well plate or dish), optionally at defined locations, foridentification of potentially therapeutic molecules. Libraries that canbe screened include, for example, small molecule libraries, siRNAlibraries, and adenoviral transfection vectors.

Such high throughput methods are therefore also applicable to predictivetoxicology. The use of cardiomyocyte cells (e.g., cardiomyoblasts,cardiomyocytes or sino-atrial nodal cells) positioned or placed(pre-seeded) on a culture dish, tube, flask, roller bottle or plate(e.g., a single multi-well plate or dish such as an 8, 16, 32, 64, 96,384 and 1536 multi-well plate or dish), optionally at defined locations,for high-throughput or high content screening using small moleculelibraries, siRNA libraries, adenoviral transfection vectors, and genebased microarray approaches can identify various therapeutic and cardiacliability targets. Such techniques also allow direct high-throughputmeasurement of cardiac intervention strategies by means of fluorescentreporter dyes and biomarkers for cell health and morphologicalphenotype, expression of fluorescent reporter proteins, various FRETapproaches and direct measurement of electrophysiological currents inlive cells.

Cardiomyocytes, as well as populations of cardiomyocytes includingenriched or selected cardiomyocytes of any developmental, maturation ordifferentiation stage thereof can be used to treat a subject in need ofincreased numbers or function of cardiomyocytes. In one embodiment, amethod for treating a subject in need of increased numbers or functionof cardiomyocytes includes administering, delivering or transplantinginto the subject a cardiomyocyte population of any developmental,maturation or differentiation stage thereof as set forth herein,optionally produced by any method as set forth herein. In anotherembodiment, a method for treating a subject in need of increased numbersor function of cardiomyocytes, includes grafting a cardiomyocytepopulation of any developmental, maturation or differentiation stagethereof as set forth herein, optionally produced by any method as setforth herein, into the heart of a subject.

The terms “subject” and “patient” are used interchangeably herein andrefer to animals, typically mammals, such as humans, non-human primates(gorilla, chimpanzee, orangutan, macaque, gibbon), domestic animals (dogand cat), farm and ranch animals (horse, cow, goat, sheep, pig),laboratory and experimental animals (mouse, rat, rabbit, guinea pig).Subjects include disease model animals (e.g., such as mice, rats andnon-human primates) for studying in vivo efficacy (e.g., a cardiacdisease or disorder animal model). Human subjects include children, forexample, newborns, infants, toddlers and teens, between the ages of 1and 5, 5 and 10 and 10 and 18 years, adults between the ages of 18 and60 years, and the elderly, for example, between the ages of 60 and 65,65 and 70 and 70 and 100 years.

Subjects include mammals (e.g., humans) in need of treatment for acardiac disease or disorder. Subjects also include those at risk ofhaving a cardiac disease or disorder. Target subjects for treatmenttherefore include those having or at risk of having a cardiac disease ordisorder.

Exemplary cardiac diseases and disorders included, but are not limitedto, atherosclerosis, stroke, congenital heart disease, congestive heartfailure, angina, myocarditis, coronary artery disease, cardiomyopathy,dilated cardiomyopathy, hypertrophic cardiomyopathy, endocarditis,myocardial infarction (Heart Attack), diastolic dysfunction,cerebrovascular disease, valve disease, high blood pressure(Hypertension), mitral valve prolapse and venous thromboembolism.

At risk subjects include those with a family history (high bloodpressure, heart disease), genetic predisposition (hypercholesterolemia),or who have suffered a previous affliction with a cardiac disease ordisorder. At risk subjects further include those with or at risk of highblood pressure or high cholesterol due to a genetic predisposition or adiet, such as high fat, or environmental exposure, such as smokers.

The doses or “amount effective” or “amount sufficient” in a method oftreatment in which it is desired to achieve a therapeutic benefit orimprovement includes, for example, any objective or subjectivealleviation or amelioration of one, several or all pathologies, adversesymptoms or complications associated with or caused by the cardiacdisease or disorder to a measurable or detectable extent. Thus, in thecase of a cardiac disease or disorder, the amount will be sufficient toprovide a therapeutic benefit to a given subject or to alleviate orameliorate a pathology, adverse symptom or complication of the cardiacdisease or disorder in a given subject. The dose may be proportionallyincreased or reduced as indicated by the status of treatment or any sideeffect(s).

In methods of treatment, a method may be practiced one or more times(e.g., 1-10, 1-5 or 1-3 times) per day, week, month, or year. Theskilled artisan will know when it is appropriate to delay or discontinueadministration. An exemplary non-limiting dosage schedule is 1-7 timesper week, for 1, 23, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more weeks, and anynumerical value or range or value within such ranges.

Of course, as is typical for any treatment or therapy, differentsubjects will exhibit different responses to treatment and some may notrespond or respond inadequately to a particular treatment protocol,regimen or process. Amounts effective or sufficient will thereforedepend at least in part upon the disorder treated (e.g., the type orseverity of the cardiac disease or disorder), the therapeutic effectdesired, as well as the individual subject (e.g., the bioavailabilitywithin the subject, gender, age, etc.) and the subject's response to thetreatment based upon genetic and epigenetic variability (e.g.,pharmacogenomics).

The invention further provides kits, including cardiomyocytes, as wellas populations of cardiomyocytes enriched or selected for anydevelopmental, maturation or differentiation stage, packaged intosuitable packaging material. In various non-limiting embodiments, a kitincludes a cardiomyocyte population that includes nodal, sino-atrial orpacemaker cells, mature contractile cardiomyocytes, immaturecardiomyocytes (cardioblasts), or a mixed population thereof. In variousaspects, a kit includes instructions for using the kit components e.g.,instructions for performing a method of the invention, such asculturing, expanding (increasing cell numbers), proliferating,differentiating, maintaining, or preserving cardiomyocytes, or acardiomyocyte cell based therapy. In various aspects, a kit includes anarticle of manufacture, for example, an article of manufacture forculturing, expanding (increasing cell numbers), proliferating,differentiating, maintaining, or preserving cardiomyocytes, such as atissue culture dish, tube, flask, roller bottle or plate (e.g., a singlemulti-well plate or dish such as an 8, 16, 32, 64, 96, 384 and 1536multi-well plate or dish). In additional various aspects, a kit includesan article of manufacture, for example, an article of manufacture fordelivering, introducing or transplanting cardiomyocytes into a subjectlocally, regionally or systemically.

The term “packaging material” refers to a physical structure housing thecomponents of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,etc.). The label or packaging insert can include appropriate writteninstructions, for example, practicing a method of the invention. Thus,in additional embodiments, a kit includes a label or packaging insertincluding instructions for practicing a method of the invention insolution, in vitro, in vivo, or ex vivo.

Instructions can therefore include instructions for practicing any ofthe methods of the invention described herein. For example,cardiomyocytes can be included in a tissue culture dish, tube, flask,roller bottle or plate (e.g., a single multi-well plate or dish such asan 8, 16, 32, 64, 96, 384 and 1536 multi-well plate or dish) togetherwith instructions for culturing, expanding (increasing cell numbers),proliferating, differentiating, maintaining, or preservingcardiomyocytes. In an additional non-limiting example, cardiomyocytescan be included in a container, pack, or dispenser together withinstructions for administration to a subject to treat a disease ordisorder or cardiac tissue, such as atherosclerosis, stroke, congenitalheart disease, congestive heart failure, angina, myocarditis, coronaryartery disease, cardiomyopathy, dilated cardiomyopathy, hypertrophiccardiomyopathy, endocarditis, myocardial infarction (Heart Attack),diastolic dysfunction, cerebrovascular disease, valve disease, highblood pressure (Hypertension), mitral valve prolapse and venousthromboembolism. Instructions may additionally include indications of asatisfactory clinical endpoint or any adverse symptoms or complicationsthat may occur, storage information, expiration date, or any informationrequired by regulatory agencies such as the Food and Drug Administrationfor use in a human subject.

The instructions may be on “printed matter,” e.g., on paper or cardboardwithin the kit, on a label affixed to the kit or packaging material, orattached to a tissue culture dish, tube, flask, roller bottle, plate(e.g., a single multi-well plate or dish such as an 8, 16, 32, 64, 96,384 and 1536 multi-well plate or dish) or vial containing a component(e.g., cardiomyocytes) of the kit. Instructions may comprise voice orvideo tape and additionally be included on a computer readable medium,such as a disk (floppy diskette or hard disk), optical CD such as CD- orDVD-ROM/RAM, magnetic tape, electrical storage media such as RAM and ROMand hybrids of these such as magnetic/optical storage media.

Invention kits can additionally include cell growth medium, bufferingagent, a preservative, or a cell stabilizing agent. Each component ofthe kit can be enclosed within an individual container or in a mixtureand all of the various containers can be within single or multiplepackages.

Cardiomyocytes, as well as populations of cardiomyocytes includingenriched or selected cardiomyocytes of any developmental, maturation ordifferentiation stage thereof can be packaged in dosage unit form foradministration and uniformity of dosage. “Dosage unit form” as usedherein refers to physically discrete units suited as unitary dosages;each unit contains a quantity of the composition in association with adesired effect. The unit dosage forms will depend on a variety offactors including, but not necessarily limited to, the particularcomposition employed, the effect to be achieved, and thepharmacodynamics and pharmacogenomics of the subject to be treated.

Cardiomyocytes as well as populations of cardiomyocytes includingenriched or selected cardiomyocytes of any developmental, maturation ordifferentiation stage thereof and methods of the invention can beincluded in or employ pharmaceutical formulations. Pharmaceuticalformulations include “pharmaceutically acceptable” and “physiologicallyacceptable” carriers, diluents or excipients. The terms“pharmaceutically acceptable” and “physiologically acceptable” mean thatthe formulation is compatible with pharmaceutical administration. Suchpharmaceutical formulations are useful for treatment of, oradministration or delivery to, or transplant into, a subject in vivo orex vivo.

Pharmaceutical formulations can be made to be compatible with aparticular local, regional or systemic administration or delivery route.Thus, pharmaceutical formulations include carriers, diluents, orexcipients suitable for administration by particular routes. Specificnon-limiting examples of routes of administration for compositions ofthe invention are parenteral, e.g., intravenous, intrarterial,intradermal, intramuscular, subcutaneous, intra-pleural, transdermal(topical), transmucosal, intra-cranial, intra-spinal, intra-ocular,rectal, oral (alimentary), mucosal administration, and any otherformulation suitable for the treatment method or administrationprotocol.

The term “contacting,” when used in reference to cells or a cell cultureor method step or treatment, means a direct or indirect interactionbetween the composition (e.g., cell or cell culture) and the otherreferenced entity. A particular example of a direct interaction isphysical interaction. A particular example of an indirect interaction iswhere a composition acts upon an intermediary molecule which in turnacts upon the referenced entity (e.g., cell or cell culture).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention relates. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed herein.

All publications, patents, Genbank accession numbers and otherreferences cited herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions,will control.

As used herein, singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to a “cardiomyocyte” includes a plurality ofcardiomyocytes, and reference to “a cell culture” can include multiplecell types of varied developmental, maturation or differentiation stagewithin the culture.

As used herein, all numerical values or numerical ranges include wholeintegers within or encompassing such ranges and fractions of the valuesor the integers within or encompassing ranges unless the context clearlyindicates otherwise. Thus, for example, reference to a range of 90-100%,includes any numerical value or range within or encompassing suchvalues, such as 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%,92.5%, etc., and any numerical range within such a range, such as90-92%, 90-95%, 95-98%, 96-98%, 99-100%, etc. In an additional example,reference to greater or less than a particular percent, e.g., greaterthan 25% means 26%, 27%, 28%, 29%, 30%, 31%, . . . etc.; and less than25% means 24%, 23%, 22%, 19%, 18%, 17%, . . . etc.

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also specificallyincludes embodiments in which particular subject matter is excluded, infull or in part, such as substances or materials, method steps andconditions, protocols, procedures, assays or analysis. Thus, even thoughthe invention is generally not expressed herein in terms of what theinvention does not include, aspects that are not expressly included inthe invention are nevertheless disclosed.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

EXAMPLES Example 1

This example is of an exemplary protocol for obtaining a mixedcardiomyocyte population.

-   -   1. One passage before differentiation, stem cells are plated in        deep dishes which can carry 1-3 cm of supernatant. Stem cells        are fed according to hES cells protocols.    -   2. Incubator set to 37-37.5° C., 2-5% O2 5-6% CO2 and 98% H2O.        Stem cells are overgrown in the original plated dishes: deep        dishes (5-10 mm of supernatant) for increasing media volume will        ensure hypoxia and enough nutriments for cellular overgrowth.        The culture will stabilize after 3-5 days of overgrowth.    -   3. Day 1-3 addition of retinoic acid (RA) 5 mM in DMSO to final        concentration of 5 μM    -   4. Day 1 to the end of the protocol. BMP4 10 ng/ml    -   5. Day 1 to the end of the protocol FGF—10 ng/ml    -   6. Replace the media daily up to day 10 and than every other day    -   7. At day 20-25 first colonies with beating clumps and typical        morphology    -   8. Day 28. The cells are incubated for 1-2 hour in a 50%        cardioplegic solution with lactic acid, protection, and absolute        hypoxia (media is degassed in vacuum and flushed with nitrogen        for 1 hour, the incubator is set for 0% oxygen or flushed        completely with nitrogen). The cardiomyocytes will switch to a        reduced anaerobic metabolism: LDH/lactic acid. The rest of the        cells will die. After 1 hour the cardioplegic solution is        removed and the cells are resuscitated with regular media.    -   9. Day 29 Death cells are removed. The purification procedure is        repeated if needed.    -   10. Day 30. The cells are incubated for 2-5 minutes with a        mixture of cardioplegic solution 50% v/v with a dissociation        enzyme (for example trypsin 0.25%). The cells are re-plated on a        fresh substrate with the same surface (1:1).    -   11. In the next days, the feeding is continued with CardioMedia        plus FGF and membrane stabilizer (Puerarin)    -   12. The culture is passaged again when confluence is reached,        using the combination of the cardioplegic solution and        dissociation enzyme.

Example 2

This example is of an exemplary protocol for obtaining early purifiedcardiomyocyte population.

-   -   1. One passage before differentiation the stem cells are plated        in deep dishes which can carry 2-3 cm of supernatant. Stem cells        are fed according to hES cells protocols.    -   2. Stem cells are overgrown in the original plated dishes until        layering of the colonies is observed (by the yellowish color on        the surface) and the media is increased in volume to ensure        hypoxia and nutriments. The culture will stabilize after 3-5        days of overgrowth    -   3. In a separate dish a pre-differentiated culture consisting of        pax6/nestin positive active dividing cells of 50,000 cells/cm2,        will incubate overnight the CardioMedia. Next day the media is        collected and filtered. The culture can be obtained from primary        culture of neural stem cells or by differentiating human        embryonic stem cells for 5-7 days in a serum free media which        contains 10 uM of retinoic acid and 5 ng/ml FGF2 added daily at        feeding.    -   4. The collected media from the neural culture is supplemented        with 10 ng/ml BMP4 and 5 ng/ml bFGF and is used to feed the        cardio cultures.    -   5. The cultures are fed every day. A mixture of some endodermal        and mostly mesodermal population is observed in 3-5 days.    -   6. Replace media every other day after day 10.    -   7. At day 28, perform the purification method described in the        above example    -   8. Dissociate cultures at day 30 and re-plate (1:1)    -   9. Feed with non-conditioned media plus FGF and membrane        stabilizers    -   10. Passage the cultures when confluents

Example 3

This example includes a description of media compositions used inexamples 1 and 2.

Cardio Media includes DMEM:F12 LO, B27 supplement, MEM-NE Aminoacids 1×,Glutamax 1×, Ascorbic acid 20 μg/ml, thyroid hormones T3/4 20 ng/ml andinsulin 10 ug/ml Cardioplegic solution includes THAM buffer(thrometamine) 0.3 Mol, KCl 10 mM, Puerarin 0.5 mM, L-MonosodiumGlutamate Monohydrate 4%, L-Monosodium Aspartate Monohydrate 4%,Trehalose 0.25%, Lactic Acid 2 mM and Ascorbic acid 20 μg/ml.Resuscitation media includes Cardio Media, Puerarin 0.5 mM, Trehalose0.25% and Superoxide dismutase.

Neural media includes DMEM:F12 high glucose, B27 Supplement 1×, MgCl 0.5mM, insulin 10 ug/ml, selenite 5 ng/ml and transferrin 20 ug/ml.

Example 4

This example includes a description of results.

Differentiation of mesodermal cells occurs shortly after some patches ofectodermal colonies are observed. The mesodermal cells typicallysurround an ectodermal cluster. The first sign of cardiomyocytedifferentiation is the formation of trabecular clusters with cellsreassembling syncitial structures. The trabecules of syncitialcardiomyocytes are radial distributed around the ectodermal center andwill start mechanical activity around days 20-25 of differentiation. Ifarrested from mechanical activity, the cardiac tissue will continuecellular division (hyperplasia). If let to continue beating, cell volumewill increase, cells will fuse in syncitial structures and willterminally differentiate (hypertrophya). (FIG. 2) Three types of cardiaccells are present in early development, nodal cells, contractilecardiomyocytes and noncontractile undifferentiated cells.

Nodal cells are immature cardiomyocytes: small cells with membrane ionic“leakage” and periodic spontaneous depolarization at threshold(pacemaker cells).

Contractile cardiomyocytes have muscular characteristics (actinfilaments and gap junctions) and are organized in syncitial mixoma likestructures.

Noncontractile, undifferentiated cells are typically actively dividing,with potential to generate nodal and contractile cells.

Cardiac cells are typically identified by detecting at least two ofcardiac markers. Particular non-limiting examples include Nkx2.5/Csx'which is first expressed in the presumptive precardiac mesoderm;transcription factors such as a GATA binding family transcription factor(GATA binding protein, such as GATA 4), MEF2, HAND, Irx, Tbx, and HRT;SRF; LIM; and alpha-actin.

Example 5

This example includes a description of characterization ofcardiomyocytes generated from human embryonic stem cells.

The human embryonic stem cell cultures were expanded in 75 cm2 cellculture flasks and underwent the same differentiation methods describedin Example 1. The cultures were then exposed to cardioplegic solutionand grown for another week. Samples were plated in imaging chambers(Nunc) and characterization was performed using antibodies against thetranscription factor NKX2.5 and a-actin. FIG. 3 illustrates the cells.

In brief, FIG. 3A illustrates extensive (hyperplasic) and homogenousgrowth of cardiomyoblasts in a 18 day old culture showing formation pftrabecular clusters (4×). FIG. 3B illustrates syncitial structures(arrows) organizing after 21-24 days of differentiation. If contractileactivity is arrested cells continue to grow in the form of high density,hyperplasic cultures (A) (20×). FIGS. 3C)-3E) illustrate cardiomyocytecultures dissociated and replated on adherent substrate at earlierstages, when NKX2.5 transcription factor is present(C) (D—Nuclearstaining with bisbenzimide; E—merged pictures). This marker ischaracteristic for myocardial and endocardial tissue (100×). FIGS.3F)-3H) illustrate more mature myocardial cells fuse and form trabecularstructures positive for a-actin (F) (G—bisbenzimide nuclear stain,H—merged picture). The actinic expression coincides with the initiationof mechanical activity. If cardioplegic media is used the syncitialagglomerates do not loose the positivity for actin, however maturationis delayed (60×). FIGS. 3I)-3K) illustrate actin labeling is persistent(I) even after enzymatic dissociation of the cultures (J—nuclear stainwith bisbenzimide, K—Merged picture (20×)). FIGS. 3L)-3N) illustratecolabeling with NKX2.5 (L) and actin (M) showing the overall expressionof the NKX2.5, while the actin is progressively expressed (N—Nuclearcounterstain (100×)).

BIBLIOGRAPHY

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1. A method for producing a culture of cardiomyocytes, comprising: A)Providing a culture of stem cells that are at least 90% confluent or thecells have overgrown to form multiple layers of cells, or proliferatingstem cells until the cells are at least 90% confluent or the cells haveovergrown to form multiple layers of cells; and B) Inducing formation ofmesoderm by contacting the overgrown stem cells with a bone morphogenicprotein (BMP) receptor ligand and an fibroblast growth factor (FGF)receptor ligand for a period of about 2 to 15 days; C) promotingcardiomyocyte formation by contacting mesoderm cells with neural cellsor endoderm cells, or a neural cell or endoderm cell conditioned culturesupernatant, for a period of about 1 to 21 days, thereby producing aculture of cardiomyocytes.
 2. The method of claim 1, wherein thecardiomyocytes comprise a population of cells comprising cells havingspontaneous and periodic electrical activity.
 3. The method of claim 2,wherein the cells having spontaneous and periodic electrical activitycomprise nodal, sino-atrial or pacemaker cells; immature cardiomyocytes(cardiomyoblasts); mature contractile cardiomyocytes; or a mixedpopulation thereof.
 4. The method of claim 1, wherein the stem cells ofstep A) comprise a cell monolayer with 100% or more confluency.
 5. Themethod of claim 1, wherein the stem cells of step A) form a multilayerculture.
 6. The method of claim 1, wherein the stem cells of step A)have a density of about 150,000 to about 250,000 cells/cm².
 7. Themethod of claim 1, wherein step B) further comprises addition of anacid.
 8. The method of claim 7, wherein the acid comprises lactic,citric, fumaric, ascorbic, folic, malic, succinic, oxalacetic, orketoglutaric acid.
 9. The method of claim 1, wherein step B) furthercomprises subjecting the mesoderm cells to hypoxia.
 10. The method ofclaim 1, wherein the stem cells, mesoderm cells or cardiomyocytes aresubjected to an oxygen concentration of about 0.5 to 2.0%.
 11. Themethod of claim 1, wherein step B) further comprises addition of anagent that inhibits cell death or apoptosis.
 12. The method of claim 11,wherein the agent comprises selenium, lithium; ascorbic acid orascorbate or superoxide dismutase (SOD).
 13. The method of claim 12,wherein the selenium comprises sodium selenite.
 14. The method of claim12, wherein the selenium is at a concentration of 1 to 20 ng/ml, or 0.5to 50 ng/ml.
 15. The method of claim 12, wherein the lithium compriseslithium carbonate.
 16. The method of claim 12, wherein the lithium is ata concentration of 1 to 20 ng/ml, or 0.5-50 ng/ml.
 17. The method ofclaim 12, wherein the ascorbic acid or ascorbate comprises sodium orcalcium ascorbate.
 18. The method of claim 12, wherein the ascorbic acidor ascorbate is at a concentration of 0.1 to 20 ug/ml, or 1 ug/ml. 19.The method of claim 12, wherein the superoxide dismutase (SOD) is at aconcentration of 1 to 100 u/ml, or 1 to 10 u/ml.
 20. The method of claim1, wherein step B) further comprises contacting the mesoderm cells witha wingless-int-1 (Wnt) family member.
 21. The method of claim 20,wherein the wingless-int-1 (Wnt) family member is Wnt-5a.
 22. The methodof claim 20, wherein the Wnt-5a is at a concentration of 1 to 10 ng/ml.23. The method of claim 1, wherein following step B), a portion of thecells express Nkx2.5/Csx marker.
 24. The method of claim 1, wherein theneural cells of step C) are produced by addition of a retinoic acidreceptor ligand to the mesoderm cells of step B).
 25. The method ofclaim 24, wherein the retinoic acid receptor ligand comprises retinoicacid.
 26. The method of claim 1, wherein the retinoic acid is at aconcentration of 1-20 mMol, or 5-10 mMol.
 27. The method of claim 1,wherein step C) comprises contact with neural cells or endoderm cellsfor about 12-48 hours.
 28. The method of claim 1, wherein the neuralcells of step C) comprise pre-differentiated or primary neural cellcultures or neural progenitor cell cultures.
 29. The method of claim 1,wherein the endoderm cells of step C) are produced by addition of a BMPreceptor ligand to the mesoderm cells of step B).
 30. The method ofclaim 1, wherein the endoderm cells of step C) comprise a primaryculture of endo-epithelial cells.
 31. The method of claim 1, whereinstep C) further comprises contacting the mesoderm cells with insulin; athyroid hormone, or an insulin-like growth factor (IGF).
 32. The methodof claim 31, wherein the insulin is at a concentration of 5 to 50 μg/ml,or 15 μg/ml.
 33. The method of claim 31, wherein the thyroid hormone isat a concentration of 1 to 40 ng/ml, or 20 ng/ml.
 34. The method ofclaim 31, wherein the thyroid hormone is T3/4.
 35. The method of claim31, wherein the IGF is IGF-1.
 36. The method of claim 31, wherein theIGF is at a concentration of 5 to 50 ng/ml, or 10 ng/ml.
 37. The methodof claim 1, wherein the cardiomyocytes express a marker selected from aGATA binding family transcription factor (GATA binding protein), MEF2(Myocyte Enhancer Factor 2), HAND (heart and neural crest derivatives),Irx, Tbx, and HRT families of transcription factors, SRF (serum responsefactor), Isl1 (Islet1), LIM (named from the Lin-11, Isl-1 and Mec-3genes) and alpha-actin.
 38. The method of claim 1, wherein the FGFreceptor ligand comprises FGF basic (FGF2, bFGF), acidic FGF (FGF1,aFGF), or a combination thereof.
 39. The method of claim 38, wherein theFGF is between about 2 to 200 ng/ml, or 5 to 20 ng/ml.
 40. The method ofclaim 1, wherein the BMP receptor ligand comprises BMP4, BMP2, BMP7 orany combination thereof.
 41. The method of claim 40, wherein the BMP isbetween about 0.1 to 100 ng/ml, or 0.5 to 10 ng/ml.
 42. The method ofclaim 1, wherein during or following steps A), B), or C) a fresh mediais added.
 43. The method of claim 42, wherein the fresh media addedduring step A) comprises a stem cell media.
 44. The method of claim 42,wherein the fresh media added during step B) comprises a cardio cellmedia.
 45. The method of claim 44, wherein the cardio cell mediacomprises a basal media and one or more of the following supplements:human albumin, essential amino acids, non essential amino acids,L-glutamine, a thyroid hormone, insulin, transferrin, ethanolamine,sodium selenite, a hydrosoluble vitamin, a liposoluble vitamin and B27supplement.
 46. The method of claim 44, wherein the basal mediacomprises DMEM, F12 or DMEM:F12.
 47. The method of claim 46, wherein theDMEM:F12 is in a ratio of about 1:1.
 48. The method of claim 1, whereina population of beating contractile cardiomyocytes is produced.
 49. Themethod of claim 48, wherein the frequency of beating is modulated bymodulating culture media pH, temperature, or a modulator drug.
 50. Themethod of claim 49, wherein the modulator drug is catecholamine, acalcium channel blocker, or potassium.
 51. The method of claim 1,further comprising step D), contacting cardiomyocytes with acardioplegic solution or treatment.
 52. The method of claim 51, whereinthe cardioplegic solution or treatment induces diastolic arrest ofbeating cardiomyocytes.
 53. The method of claim 51, wherein thecardioplegic solution or treatment induces reduces energy requirement ofbeating cardiomyocytes.
 54. The method of claim 51, wherein thecardioplegic solution or treatment inhibits hypoxia induced damage ofbeating cardiomyocytes.
 55. The method of claim 51, wherein thecardioplegic solution comprises a mixture of one or more of: KCl (10-20mM); MgCl₂ (10 mM).
 56. The method of claim 51, wherein the cardioplegicsolution further comprises one or more of CaCl2 (1.2 mM); puerarin (0.5mM), or Nifedipine (1-10 uM).
 57. The method of claim 51, wherein thecardioplegic solution comprises a tris(hydroxymethyl)aminomethane orHanks balanced salt solution adjusted to a pH of approximately 8.6. 58.The method of claim 51, wherein the cardioplegic solution furthercomprises a mixture of L-Monosodium Glutamate Monohydrate andL-Monosodium Aspartate Monohydrate.
 59. The method of claim 58, whereinthe mixture comprises about 4.277% of L-Monosodium Glutamate Monohydrateand about 3.923% of L-Monosodium Aspartate Monohydrate.
 60. The methodof claim 1 or 51, further comprising steps D) or E), isolating immaturecardiomyocytes (cardioblasts) prior to beating.
 61. The method of claim1 or 51, further comprising steps D) or E), isolating mature contractilecardiomyocytes.
 62. The method of claim 1 or 51, further comprisingsteps D) or E), preserving, freezing or storing the cardiomyocytes. 63.The method of claim 1 or 51, further comprising steps D) or E),enriching or selecting for cardiomyocytes.
 64. The method of claim 63,wherein enriching or selecting for cardiomyocytes comprises subjectingthe cells to a treatment that requires anaerobic metabolism so thatcells unable to survive by anaerobic metabolism senesce or die.
 65. Themethod of claim 63, wherein enriching or selecting for cardiomyocytescomprises exposure to hypoxia, contact with lactic acid or contact witha cardioplegic solution or treatment.
 66. The method of claim 63,wherein enriching or selecting for cardiomyocytes comprises identifyingcardiomyocytes and removing the identified cardiomyocytes.
 67. Themethod of claims 60 to 63, further comprising steps E) or F), recoveringthe enriched or selected cardiomyocytes.
 68. The method of claims 60 to63, further comprising steps E) or F), recovering the enriched orselected cardiomyocytes and distributing the enriched or selectedcardiomyocytes in a cell culture dish, plate, vial, tube, flask orbottle.
 69. The method of claims 60 to 63, further comprising steps E)or F), recovering the enriched or selected cardiomyocytes and freezingthe enriched or selected cardiomyocytes in a solution.
 70. Acardiomyocyte population produced by the method of any of claims 1 to69.
 71. A cardiomyocyte population comprising nodal, sino-atrial orpacemaker cells, mature contractile cardiomyocytes, immaturecardiomyocytes (cardioblasts), or a mixed population thereof produced bythe method of any of claims 1 to
 69. 72. A kit comprising acardiomyocyte population produced by the method of any of claims 1 to69.
 73. A method for identifying a cardioactive agent, comprising: A)contacting a cardiomyocyte population produced by the method of any ofclaims 1 to 69 with a test agent; and B) determining if the test agentmodulates an activity or function of cardiomyocytes within thepopulation, wherein modulating an activity or function of cardiomyocyteswithin the population identifies the test agent as a cardioactive agent.74. The method of claim 73, wherein the activity or function comprisescontraction or beating.
 75. The method of claim 73, wherein the activityor function comprises production of a metabolic product or intracellularenzyme.
 76. The method of claim 75, wherein the metabolic product is oneor more of urea, creatine or CO2.
 77. The method of claim 75, whereinthe intracellular enzyme is one or more of lactate dehydrogenase,creatine phosphokinase (CPK), creatine kinase (CK) or troponin.
 78. Themethod of claim 73, wherein the activity or function comprises cellularapoptosis, necrosis, death; or dedifferentiation, maturation, division.79. A method for treating a subject in need of increased numbers orfunction of cardiomyocytes, comprising transplanting into the subject acardiomyocyte population produced by the method of any of claims 1 to69.
 80. A method for treating a subject in need of increased numbers orfunction of cardiomyocytes, comprising grafting a cardiomyocytepopulation produced by the method of any of claims 1 to 69 into theheart of a subject.
 81. A cell culture of immature cardiomyocytes(cardiomyblasts), wherein 50% or more of said culture comprises immaturecardiomyocyte (cardiomyblast) cells, and wherein no more than about 30%of said cells beat or contract.
 82. The cell culture of claim 81,wherein 60% or more of said culture comprises immature cardiomyocyte(cardiomyblast) cells.
 83. The cell culture of claim 81, wherein 70% ormore of said culture comprises immature cardiomyocyte (cardiomyblast)cells.
 84. The cell culture of claim 81, wherein 80% or more of saidculture comprises immature cardiomyocyte (cardiomyblast) cells.
 85. Thecell culture of claim 81, wherein 90% or more of said culture comprisesimmature cardiomyocyte (cardiomyblast) cells.
 86. The cell culture ofclaim 81, wherein no more than about 25% of said cells beat or contract.87. The cell culture of claim 81, wherein no more than about 20% of saidcells beat or contract.
 88. The cell culture of claim 81, wherein nomore than about 15% of said cells beat or contract.
 89. The cell cultureof claim 81, wherein no more than about 10% of said cells beat orcontract.
 90. The cell culture of claim 81, wherein no more than about5% of said cells beat or contract.
 91. A cell culture of immaturecardiomyocytes (cardiomyblasts), wherein 50% or more of said cellculture comprises immature cardiomyocyte (cardiomyblast) cells, andwherein 75% or more of said immature cardiomyocyte (cardiomyblast) cellssurvive anaerobic conditions for 30, 60 or more minutes.
 92. A cellculture of immature cardiomyocytes (cardiomyblasts), wherein 50% or moreof said culture comprises immature cardiomyocyte (cardiomyblast) cells,and wherein no more than about 25% of said immature cardiomyocyte(cardiomyblast) cells die when subjected to anaerobic conditions for 30,60 or more minutes.
 93. The cell culture of claim 92, wherein no morethan about 20% of said immature cardiomyocyte (cardiomyblast) cells diewhen subjected to anaerobic conditions for 30, 60 or more minutes. 94.The cell culture of claim 92, wherein no more than about 15% of saidimmature cardiomyocyte (cardiomyblast) cells die when subjected toanaerobic conditions for 30, 60 or more minutes.
 95. The cell culture ofclaim 92, wherein no more than about 10% of said immature cardiomyocyte(cardiomyblast) cells die when subjected to anaerobic conditions for 30,60 or more minutes.
 96. The cell culture of claim 92, wherein no morethan about 5% of said immature cardiomyocyte (cardiomyblast) cells diewhen subjected to anaerobic conditions for 30, 60 or more minutes. 97.The cell culture of claim 92, wherein said anaerobic condition comprisesan atmosphere of 0%-2% oxygen.
 98. The cell culture of claim 92, whereinthe immature cardiomyocyte (cardiomyblast) cells proliferate.
 99. Thecell culture of claim 92, wherein no more than about 30% of saidimmature cardiomyocyte (cardiomyblast) cells exhibit a functionassociated with contractile cardiomyocytes.
 100. The cell culture of anyof claim 81, 91 or 92, further comprising a substrate to which the cellsare attached.
 101. The cell culture of claim 100, wherein the substratecomprises a multiwell plate or dish having disposed thereon thecardiomyocyte (cardiomyblast) cells in one or more of said wells.
 102. Akit comprising the immature cardiomyocyte (cardiomyblast) cells of anyof claim 81, 91 or 92.